Real-time Imaging of NADPH Oxidase Activity in Living Cells Using a Novel Fluorescent Protein Reporter

PLoS One. 2013 May 21;8(5):e63989. doi: 10.1371/journal.pone.0063989. Print 2013.

Abstract

Production of reactive oxygen species (ROS) has been implicated in the pathology of many conditions, including cardiovascular, inflammatory and degenerative diseases, aging, muscular dystrophy, and muscle fatigue. NADPH oxidases (Nox) have recently gained attention as an important source of ROS involved in redox signaling. However, our knowledge of the source of ROS has been limited by the relatively impoverished array of tools available to study them and the limitations of all imaging probes to provide meaningful spatial resolution. By linking redox-sensitive GFP (roGFP) to the Nox organizer protein, p47(phox), we have developed a redox sensitive protein to specifically assess Nox activity (p47-roGFP). Stimulation of murine macrophages with endotoxin resulted in rapid, reversible oxidation of p47-roGFP. In murine skeletal muscle, both passive stretch and repetitive electrical stimulation resulted in oxidation of p47-roGFP. The oxidation of p47-roGFP in both macrophages and skeletal muscle was blocked by a Nox specific peptide inhibitor. Furthermore, expression of p47-roGFP in p47(phox) deficient cells restored Nox activity. As Nox has been linked to pathological redox signaling, our newly developed Nox biosensor will allow for the direct assessment of Nox activity and the development of therapeutic Nox inhibitors.

Publication types

  • Research Support, N.I.H., Extramural
  • Research Support, Non-U.S. Gov't

MeSH terms

  • Animals
  • Biosensing Techniques
  • Cell Survival / drug effects
  • Computer Systems*
  • Electric Stimulation
  • Extracellular Space / drug effects
  • Extracellular Space / metabolism
  • Fluorescence
  • Genes, Reporter*
  • Green Fluorescent Proteins / metabolism*
  • Humans
  • In Vitro Techniques
  • Lipopolysaccharides / pharmacology
  • Macrophage Activation / drug effects
  • Membrane Glycoproteins / deficiency
  • Membrane Glycoproteins / metabolism*
  • Mice
  • Molecular Imaging / methods*
  • Muscle Fibers, Skeletal / drug effects
  • Muscle Fibers, Skeletal / enzymology
  • NADPH Oxidase 2
  • NADPH Oxidases / deficiency
  • NADPH Oxidases / metabolism*
  • Oxidation-Reduction / drug effects
  • Reactive Oxygen Species / metabolism
  • Time Factors

Substances

  • Lipopolysaccharides
  • Membrane Glycoproteins
  • Reactive Oxygen Species
  • Green Fluorescent Proteins
  • Cybb protein, mouse
  • NADPH Oxidase 2
  • NADPH Oxidases
  • neutrophil cytosolic factor 1